1. Field of the Invention
The present invention relates generally to the field of neoplastic disease treatment. More particularly, it concerns the intravenous administration of highly concentrated solutions of phenylacetylglutamine and phenylacetylisoglutamine, or phenylacetylglutamine and phenylacetate, or salts or derivatives thereof, at high infusion rates and high dosage levels.
2. Description of Related Art
Research on growth factors and growth inhibitors during the last thirty years indicates the possible existence of a defense system of the human body complementary to the immune system. This defense system of differentiation inducers and regulators of oncogene and tumor-suppressor-gene expression may be termed a “biochemical defense system” or “BDS.” Whereas the main purpose of the immune system is protection of the body against external invasion, the main purpose of the BDS is protection of the body against defective cells. Human neoplastic diseases (cancers, malignant and benign tumors) are examples of diseases that can be combated by the BDS. One class of compounds that provide components of the BDS are naturally-occurring amino acid analogs and carboxylic acids.
Though not to be bound by theory, the mechanism of defense against cancers by naturally-occurring amino acid analogs can be induction of differentiation, conjugation of glutamine to inhibit growth of cancerous cells, downregulation of oncogenes such as ras, or upregulation of detoxification genes such as GSTP1 and GSTM1 and tumor suppressor genes such as p53, retinoblastoma gene, and neurofibromatosis gene type 1, possibly by decreasing methylation of hypermethylated genes. Regardless of the detailed mechanism of action, naturally-occurring amino acid analogs are known to induce abnormal cells to undergo terminal differentiation and die through programmed cell death. Unlike necrosis associated with chemotherapy or radiation therapy, dying cells are gradually eliminated and replaced by normal cells, leading to organ healing and reconstruction of function.
The study of naturally-occurring amino acid analogs as potential anti-cancer agents, hereinafter generally “antineoplastons,” began in 1967 with the observation of significant deficiencies in the serum peptide content of cancer patients. During the 1980's, the isolation of antineoplaston fractions from human urine and the use of these fractions in the treatment of human cancers were taught by Burzynski, U.S. Pat. No. 4,470,970, the entire specification of which is incorporated by reference herein. Among the compositions put forth as cancer treatments were (a) 3-phenylacetylamino-2,6-piperidinedione, and (b) a mixture of sodium phenylacetate and phenylacetylglutamine in a 4:1 ratio by mass. Composition (b) may hereinafter be referred to as “antineoplaston AS2-1” or simply “AS-2-1.” 3-phenylacetylamino-2,6-piperidinedione was discovered to hydrolyze during treatment with sodium hydroxide upon dissolving and neutralization to phenylacetylglutamine and phenylacetylisoglutamine in a 4:1 ratio.
Formulations of the above compositions were prepared and had successful preclinical activity. 3-phenylacetylamino-2,6-piperidinedione produced a cytostatic effect on cultured human breast cancer cell line MDA-MB-231. Dose-dependent inhibition of the growth curves of cell lines KMCH-1, KYN-1, and KIM-1; rat Nb2 lymphoma; and human colon adenocarcinoma was also observed upon administration of 3-phenylacetylamino-2,6-piperidinedione.
In vivo experiments were performed in which 3phenylacetylamino-2,6-piperidinedione, or A10, was administered to mice implanted with S180 cells or R-27 human breast cancer cells. In the S180 experiment, cAMP levels in the livers and tumors of treated mice were significantly elevated relative to control mice after administration of 3-phenylacetylamino-2,6-piperidinedione. In the R-27 experiment, 3H-TdR uptake inhibition and growth curve inhibition were observed after injection of A10.
AS-2-1 or phenylacetic acid produced dose-dependent growth inhibition in breast carcinoma cell line HBL-100 and Ki-1, and also promoted terminal differentiation or phenotypic reversion in cell lines of human promyelocytic leukemia HL-60, chronic lymphocytic leukemia, neuroblastoma, murine fibrosarcoma V7T, hormonally refractory prostate adenocarcinoma PC3, astrocytoma, medulloblastoma, malignant melanoma and ovarian carcinoma. AS2-1 or phenylacetic acid caused adipocyte conversion in cultured premalignant mesenchymal C3H 10T1/2 cells and enhanced hemoglobin production in K562 erythroleukemia cells. Further, and in distinction to then-current standard chemotherapeutic agents such as 5-aza-2-deoxycitidine, phenylacetic acid did not cause tumor progression in premalignant C3H 10T1/2 cells.
Preclinical toxicology studies determined that the LD50 for A10 in mice was 10.33 g/kg/day. Autopsy of animals which died revealed generalized congestion of the viscera, pulmonary edema, and hemorrhagic changes in the alveoli. In autopsy, surviving test animals were identical to control animals. Chronic toxicity studies revealed no negative effects after 180 days.
The LD50 for AS2-1 in mice was 2.83 g/kg/day. Autopsy of animals which died revealed generalized congestion of the viscera, pulmonary edema, and hemorrhagic changes in the alveoli, as well as Tardieu's spots and congestion of the thymus. Chronic toxicity studies using up to 1.11 g/kg/day revealed no negative effects after 365 days.
A10 and AS2-1 were observed to be non-mutagenic by the Ames method, and A10 was observed to be non-teratogenic in rat fetuses.
A noteworthy point regarding the toxicology studies is that phenylacetylglutamine, a component of AS2-1 and also a breakdown product of 3-phenylacetylamino-2,6-piperidinedione, is not normally found in mice but is normally found in humans. This suggests that humans might exhibit greater tolerance of both A10 and AS2-1 than do mice, and thus higher doses of both compositions might be possible in humans. This suggestion is accurate as will be shown below.
In human toxicity studies in Phase I clinical trials, intravenous administration of A10 at dosages up to 2.21 g/kg/day was associated with minimal side effects, including febrile reaction, muscle and joint pain, muscle contraction in the throat, abdominal pain of short duration, and single incidences of nausea, dizziness, and headache (Drugs Exptl Clin Res 1986, 12 Suppl 1, 47-55).
Oral administration of AS2-1 at dosages up to 238 mg/kg/day was associated with a temporary mild decrease in white blood cell count in one patient. Injection of AS2-1 at dosages up to 160 mg/kg/day was associated with minimal side effects, including slight nausea and vomiting, allergic skin reaction, moderate elevation of blood pressure, febrile reaction, mild decrease in white blood cell count, (one patient each) and mild electrolyte imbalance in three patients.
Clinical trials determined that 3-phenylacetylamino-2,6-piperidinedione, A10, and AS2-1 were effective in treating cancer. Burzynski et al. (Drugs Exptl. Clin. Res. 12 Suppl. 1, 25-35 (1986)) reported that an intravenous solution of antineoplaston AS2-1 (100 mg/mL active ingredients) was injected into patients at dosages of not more than 0.16 g/kg/day. Of 21 cases of neoplastic disease, observed were six complete remissions, two partial remissions, seven stabilizations, and six cases of progressive disease.
Phase II clinical trials were conducted wherein patients suffering from astrocytomas were infused with A10 (100 mg/mL) at dosage levels of from 0.5 to 1.3 g/kg/day or with AS2-1 (100 mg/mL) at dosage levels of from 0.2 to 0.5 g/kg/day for from 67 to 706 days (in: Recent Advances in Chemotherapy, Adam, D., ed. Munich: Futuramed, 1992). Of 20 patients, four experienced complete responses, two experienced partial responses, ten experienced stabilizations, and four experienced progressive disease.
In Samid, U.S. Pat. No. 5,605,930 (the entire content of which is incorporated by reference herein), sodium phenylacetate alone was used in treating human cancers, and was administered in dosages of not more than 0.3 g/kg/day. However, a number of shortcomings of the low concentrations, flow rates, and dosages of the intravenous solutions were observed.
First, Burzynski et al. (Drugs Exptl. Clin. Res. 12 Suppl. 1, 11-16 (1986)) reported complete colony reduction of HBL-100 and Ki No. 1 tumor cell lines with 5.0 mg/mL of either phenylacetic acid or antineoplaston AS2-1. Similarly, cytostasis was observed for human breast carcinoma cell line MDA-MB-231 using concentrations of 3-phenylacetylamino-2,6-piperidinedione of 2.0 mg/mL and AS2-1 of 3.0 mg/mL. However, 3-phenylacetylamino-2,6-piperidinedione is poorly soluble in water, and when orally administered to rats the peak plasma level is approximately 0.2 mg/mL, roughly 10-fold less than the cytostatic concentration observed in tissue culture experiments. Under typical administration regimes of antineoplaston AS2-1, the peak plasma levels of phenylacetic acid are approximately 0.43 mg/mL, roughly 7-fold less than the cytostatic concentration observed in tissue culture experiments. Also, both 3-phenylacetylamino-2,6-piperidinedione, its hydrolysis products, and AS2-1 are rapidly cleared in vivo.
Also, during uptake of antineoplastons by tumor tissue, a concentration gradient forms between the outside of the tumor tissue, at which the concentration of antineoplaston will be equal to the plasma concentration, and a point or points in the interior of the tumor tissue, at which the concentration of antineoplaston will be at a minimum, and may be zero. Relatively low plasma concentrations of anti-cancer agents therefore lead to some inner portion of the tumor tissue avoiding significant uptake of the anti-cancer agent and remaining in its cancerous state.
Second, administration of a solution comprising the hydrolysis products of 3-phenylacetylamino-2,6-piperidinedione at low infusion rates of from 2.5 mL/h to 84 mL/h frequently results in an elevation in levels of waste products in plasma. An exemplary waste product so elevated is uric acid. This elevation interferes with treatment by requiring either a decrease in the dose or an interruption in the treatment to administer additional drugs, for example, Allopurinol, to decrease the level of the waste product, for example, uric acid.
Therefore, it is desirable to have intravenous formulations of pharmaceutical compositions of amino acid analogs with anti-cancer activity wherein the intravenous formulations provide high plasma concentrations of the active ingredient or ingredients in order to fully penetrate tumors with effective amounts of the active ingredient or ingredients. It is also desirable that such intravenous formulations do not lead to elevated levels of waste products in plasma.